Mössbauer study of Clostridium pasteurianum hydrogenase II. Evidence for a novel three-iron cluster.

Hydrogenase II contains two iron-sulfur clusters, one of the [4Fe-4S] type and one of unknown structure with unusual spectral properties (H-cluster). Using Mössbauer spectroscopy we have studied the H-cluster under a variety of conditions. In the reduced state the cluster exhibits, in zero magnetic field, spectra with the typical 2:1 quadrupole pattern of reduced [3Fe-4S] clusters. However, whereas the latter are paramagnetic (S = 2) the H-cluster is diamagnetic (S = 0). Upon oxidation and exposure to CO the H-cluster exhibits an S = 1/2 EPR spectrum with g values at 2.03, 2.02, and 2.00. In this state, the Mössbauer spectra reveal two cluster subsites with magnetic hyperfine coupling constants AI = +26.5 MHz and AII = -30 MHz. ENDOR data obtained by Hoffman and co-workers (Telser, J., Benecky, M. J., Adams, M. W. W., Mortenson, L. E., and Hoffman, B. M. (1986) J. Biol. Chem. 261, 13536-13541) show a 57Fe resonance at AIII approximately equal to 9.5 MHz. Analysis of the Mössbauer spectra shows that this resonance represents one iron site. Our studies of the reduced and CO-bound oxidized states of hydrogenase II suggest that the H-cluster contains three iron atoms. The data obtained for the oxidized H-cluster suggest a novel type of 3-Fe cluster and bear little resemblance to those reported for oxidized [3Fe-4S] clusters with g = 2.01 EPR signals. In the reduced sample the [4Fe-4S]1+ cluster appears to occur in a mixture of two distinct electronic states.